Stringent and relaxed control of phospholipid metabolism in Escherichia coli.

Abstract Stringent cells of Escherichia coli (rel+) cease growth and protein, RNA, and lipid synthesis when deprived of a required amino acid; relaxed cells (rel-) cease growth and protein synthesis but continue lipid synthesis when deprived of a required amino acid, as assayed by [14C]acetate incorporation. When phospholipid synthesis was assayed by using 32Pi, some net synthesis does occur, but the majority of the incorporation is a consequence of rapid breakdown and resynthesis (turnover) of phosphatidylethanolamine, normally a stable component of the cell envelope.

Prom the Department of Chemistry and Geology, Clemson University, Clemson, South Carolina 29631 SUMMARY Stringent cells of Escherichia co2i (reZ+) cease growth and protein, RNA, and lipid synthesis when deprived of a required amino acid; relaxed cells (re2-) cease growth and protein synthesis but continue lipid synthesis when deprived of a required amino acid, as assayed by [14C]acetate incorporation.
When phospholipid synthesis was assayed by using s2Pi, some net synthesis does occur, but the majority of the incorporation is a consequence of rapid breakdown and resynthesis (turnover) of phosphatidylethanolamine, normally a stable component of the cell envelope.
When growing bacteria are deprived of a required amino acid they stop growing and quit making protein and stable RNA. The requirement for adequate supplies of amino acids for protein synthesis is apparent; however, the biochemical basis of the relationship between amino acid starvation and stable RNA synthesis is not understood (I).
Organisms which are relaxed (Tel-) (i.e. with a genetic lesion in the ribonucleic acid control (RC) gene) in contrast to wild type or stringent (Tel+) organisms synthesize ribosomal and transfer RNA at nearly the normal rate (2) although growth and protein synthesis have essentially stopped.
Although a great deal of attention has centered on the effects of stringent and relaxed control of RNA synthesis (3, 4; see general reviews, 5, 6), a variety of other processes are apparently also affected, including translation of RNA into protein (7), phage DNA synthesis (8), nucleotide metabolism (g-12), glucose transport (13), and lipid biosynthesis (14). We wished to characterize the control exerted by the RC gene under these conditions on the biosynthesis of phospholipidsin Escherichia coli, since these * This work was supported by funds from the Department of Chemistry and Geology and by Grant GB-30072 to G. L. P., from the Metabolic Biology Section of the National Science Foundation. Preliminary reports of this work were presented to the Southeastern Regional American Chemical Society in Nashville, Tenn., November 5, 1971, and to the Federation of American Societies for Experimental Biology in Atlantic City, N. J., April 11, 1972 (Abstract No. 20716).
i These studies were taken from a thesis submitted by Norma G.'Golden to Clemson University in partial fulfillment of the requirements for the Masters Degree in Chemistry.
5 To whom reprint requests should be addressed.
are the major lipids (95%) of this organism (15) and since very little information exists on regulation of phospholipid biosynthesis in general (16).
We have verified the earlier (14) report of relaxed and stringent control of lipid synthesis.
Other workers (17) have questioned the stringent control of lipid synthesis but these differences now seem to have been the result of decreased aeration.
A primary difference in phospholipid metabolism between stringent and relaxed organisms seems to be a marked turnover of phosphatidylethanolamine, the major, normally stable phospholipid in these organisms, in amino acid-starved, relaxed cells.
All are K-12 strains and are believed to be isogenic except for a small region of the chromosome between Minute 53 and 54 on the Taylor-Trotter map which includes the rel locus W.
All isotopes were obtained from New England Nuclear Corp. Lipid standards were obtained as a gift from Dr. J. E. Cronan or from Supelco, Inc. Silica Gel G w as obtained from Brinkmann Instrument Co.; silica gel-loaded paper (Whatman SG-81) was from Reeve Angel Co. The chloroform was of United States Patent grade. All other chemicals were reagent grade. All solvents were dried over molecular sieve (Linde 4A) before use.
Growth Conditions-In experiments where radioactive phosphate was not employed, the cells were grown in phosphate-rich Medium E (19). Glucose and, in one set of experiments, succinate and ribose were used as the carbon source by sterilizing each separately and adding to a final concentration of 0.2, 0.4, and 0.3%, respectively.
Amino acids were added to a final concentration of 10F3 M, and thiamine was added to a final concentration of 0.5 pg per ml. The phosphate-poor medium (Trismaleate buffer) used when the cells were to be labeled with radioactive phosphate is identical with phosphate-rich Medium E, except that the phosphates are replaced by 5.80 g of maleic acid, 6.06 g of 2-amino-2-(hydroxymethyl)-1,3-propanediol (Tris), and 0.746 g of KC1 adjusted to pH 7.4 (19).
All experiments are carried out at 37" in 25 to 35 ml of medium in 250.ml Erylenmeyer flasks in a New Brunswick rotary incubator shaker at 180 rpm. Growth was measured by following the turbidity at 620 nm with a Bausch and Lomb Spectronic 600 spectrophotometer. Amino acid starvation was induced by resuspending the culture in medium lacking only the required amino acid. Radioactive isotopes employed were added to the culture after the nutritional components but before the cells.
Assay of Phospholipids-A modification of the Bligh and Dyer (20) extraction procedure as suggested by Ames (21) for E. coli was employed. The cells were extracted for 30 min at room temperature, and the chloroform layers obtained after separation of the phases were evaporated to dryness under a stream of nitrogen. The lipid was taken up in a small volume of chloroformmethanol (2 : 1) and stored at 5".
Commercial silica gel-loaded paper was used for the separation of phospholipids. The paper was cut in strips (21 x 31 cm), and their ends were stapled together to form cylinders. For activation of the paper, the chromatograms were first developed in dry acetone. The chromatographs were removed from the acetone tank, and the lipid sample was immediately applied and then developed in a solvent system (22) containing chloroform, methanol, and water (65:25:4) at room temperature for 134 to 2 hours. The separations were good and quite reproducible ( Fig.  1).
Phospholipids were identified by chromatography with com-mercial standards and with the phospholipids of defined composition from E. coli strain K-12 (21, 23). Radioactive lipids were located by autoradiography using Kodak No-Screen x-ray film. About 5000 cpm of BP can easily be detected after a 24-hour exposure to the film. Quantitative estimation of radioactivity was obtained by cutting out the specific area from the paper chromatogram and assaying for radioactivity in a liquid scintillation counter. Our recoveries were 99% when the spots were cut from the silica gel-loaded paper and counted directly. Total CHCla-CHSOH-soluble phospholipid phosphate was determined by the method of Ames by using the Mg(NO& ashing procedure (24).

Assay of Phospholipid
Synthesis and Turnover-Incorporation of [l%]acetate into lipid was measured by the method of Sokawa et al. (14). We also used the extraction procedure described below (21) with equivalent results.
Incorporation of azPi into phospholipids was assayed as follows. Cells grown in phosphate-rich medium with all of the nutritional requirements for normal growth were harvested in the log phase of growth by centrifugation at room temperature. The cell pellet was washed with phosphate-free medium at 37" and centrifuged again. The cells were resuspended in medium at 37" to which phosphate (0.3 mM final concentration) and the Carrier-free 32Pi was added (0.1 PCi per ml), and the cultures were incubated at 37" with shaking.
,\liquots (4 ml) were removed at various time intervals, extracted by the procedure described earlier (al), and analyzed for V rontent.
The 32Pi specific activity of the medium was determined for every experiment.
h portion (10 ~1) of the medium and 990 ~1 of water were added t,o a counting vial. The samples were then assayed for radioactivity.
The phosphate concent'ration of the medium was 0.3 mM.
The rate of turnover of pl~ospl~olipids was assayed by a dilution technique.
Cells were first fully labclcd by growing in the presence of 32Pi (0.1 PCi l)rr ml) for 7 to 12 hours. The cells were resuspended in medium at 37" containing 0.3 mhf unlabeled phosphate, the necessary carbon source, and vitamins, with or without the required amino acid. The loss of radioactivity from the labeled lipid pool was followed by removing IO-In1 aliquots at various time intervals, cxstracting and analyzing as previously described.
The rate of breakdown and renewal of phospholipids could be most accurately determined by using a dual labeling technique with 33Pi and 32Pi. These experiments were carried out identically to the dilution experiment.
The cells were first fully labeled with 33Pi (0.07 PCi per ml) and then resuspended in rnediurn containing 0.3 mM phosphate and a suitable concentration of 32Pi. (The 32Pi specific activity was adjusted so that the net incorporation of radioactivity into nongrowing cells was comparable to that in growing cells. This adjustment was necessary to get optimal assay of 32P in the presence of 331'.) Radioassay-Ratlioac:tivit?-was determined using a Packard Tri-Carb liquid scintillation spectrometer, model 3320. Rqueous solutions were counted in 10 ml of Triton-X counting solution (333 ml of Triton-X-100, 667 ml of scintillation grade toluene, 0.1 g of 1,4 bis[2~(5~phcnyloxazolyl)]benzene (POPOP), and 5.5 g of 2,5-diphenyloxazole (1'1'0) (25) containing a total of 1 ml of water.
Chloroform extracts were counted by removing an aliquot and pipetting directly into an empty scintillation vial. The samples were evaporated to dryness under a stream of nitrogen and counted in 10 ml of 'Triton-X counting solution. Suitable gain settings for assaying32P and 33P were chosen in the Triton-X counting solution, using an Engberg plot as a guide (26). An efficiency of 98% for Y' and 24% for 32P could be obtained in one channel.
In a second channel, 2% V efficiency and 76% 32P efliciency were obtained by suitable adjustment of the window settings.
32P was assayed in 1 : 1 (v/v) chloroform-methanol (the window settings were 1000 to m at 45% gain) with an efficiency of 55y0. Since the efficiency for 331' in this solution was approximately 0% (27), we could check the H333P04 supplied for 32P. WC observed less than 5y0 321' dpm in the H~~~1'04 used in the dual label esperiments.
Other Assays-Protein biosynthesis was assayed by the method of Ryfield and Scherbaum (28) by using 0.02 &i of [G-3H]isoleucine per ml. RNA biosynthesis was determined the same way by using 0.1 MCI of [6-3H]uracil per ml, but in the presence of 10 pg of carrier uracil per ml.

RNA, Protein, and Lipid Biosynthesis in Relaxed and Stringent Cells-Relaxed
and stringent control can be demonstrated by resuspending an exponentially growing culture of cells auxotrophic for one or more amino acids in fresh, complete medium in the presence or absence of a required amino acid. &say for RNA synthcais in the prcscnce or abse~lcc of that amino acid defines t,he phenotype; relaxed cells synthesize RNA in the abscncc of tllc required amino acid, stringent cells do not. Lack of both growth and protein biosynthesis in the abscncc of the required arnino acid and wild type growth and synthesis in the presence of the required amino a&l are useful controls. Lipid biosynthesis was followed by cletermiiiiug the amount of ['%'Iacsetate incorporated into lipid.
The results of typical cusp& merits are given in Figs. 2 to 4.
When stringent cells are deprived of a required amino acid, lipid biosynthesis (Fig. 4) proceeds at a minin~al rate in stringent cells (significant incorporation does o~ur; coiltrast with RN.1 synthesis in Fig. 3) and at twice the minimal rate in relaxed ~11s. The rate of lipid biosynthesis attained by amino acid-dellrived rclascd cells is, howcvcr, only 25% that attained by growing cells. This is t,hc same result obtained by Sokawa et al. (14) with this organism, and is idcntiral with the results which can bc obtained with another stringcat and relaxed pair, PA-1 and PA-2.
Incorporation 01 32Pi into Phospholipid in Relaxed and Stringent Cells--[14C]i\cetatc can bc incorporated into a variety of mctabolites iu Ei:. coli, and the rate of that incorporation may be a complex function of the growth conditions. However, if the relaxed control were a general control of l~l~osl~holipid biosynthc~is one should be able to ob$crve rclascd and stringent control of 321'i incorporation into phospholipid. Tlris general observation is correct for at least two different pairs of rclased and stringent organisms (Figs. 5 and 6). In these figures the incorporation of %I' into lipid is normalized to a constant cell mass, emphasizing the rate of phospholipid synthesis in the relaxed cells; t'he incorporation is nearly equal to that of exponentially growing cells. Essentially identical results are obtained when 32P incorporation is assayed using succinate (Fig. 7) or ribose as the carbon and energy source, although the total incorporation II-as less than when the cells are labeled in the presence of glucose as carbou and energy source.
Phospholipid Turnover in Relaxed and Stringent Cells-The 321' incorporation into l~l~ospholil~id may be a result of synthesis leading to a net increase in phospholipid content per unit of cell mass or, if the phospholipids are degraded as rapidly as they are formed, 321' incorporation rnay take place with no net' increase in the phospholipid content of the cells. Perhaps the most direct way to measure a net increase in phospholipid content is to assay total extractable lipid phosphate directly.
The results of such a determination on cells grown and sampled just as described for experiments employing [Wlacetate or 321'i are shown in Fiv. 8. These results are comparable to those obtained with [W]ace?ate (Fig. 4) and support the strirrgcnt and relaxed control of phospholipid.
If these data were represcntcd as total lipid phosphate per uuit of cell mass, relaxed cells, when starved for a required amino acid, in contrast to stringent cells would be found to increase in total pl~osl~l~olil~id content per unit cell mass. The net increase in phospholipid in relaxed amino acid-deprived cells was ruuch less than anticipated from 321' in corporation data (Figs. 5 and 6). Thus the incorporation of 321) into the lipids of stringent amino acid-deprived cells must be a consequence of turnover since there is no net increase of total phosl~holil~id per unit cell mass.
The degradation of l~hospholipids can be assayed directly by observing the loss of 3zJ) from the phospholipids of cells that are fully labeled frorn growth on 321'i and resuspended in unlabeled phosphate-containing medium.
We found in these experirncnts, carried out just as before, that the phospholipids of relaxed deprived cells are apparently degratletl at a much more rapid rate    than relaxed exponentially growing cells or stringent deprived but this behavior is relatively not much different from growing cells. cells (Fig. 13) or stringent amino acid deprived cells (Fig. 14). A dual label experiment with cells grown under conditions identical with the above compared the extent of synthesis and degradation simultaneously. The cells were grown for 10 or more generations in medium containing 33Pi of known specific act'ivitg, thus ensuring that the phospholipid phosphodiester phosphate should be of essentially the same specific activity as the medium.
When either relaxed or stringent cells were resuspended in the absence of a required amino acid, nearly half of the radioactivity of cardiolipin was lost (Figs. 15 to 17). The remaining portion seems to be relatively stable. Cardiolipin was actively resynthesized in the relaxed amino acid-deprived cells (Fig. 15).
We fractionated these fully labeled phospholipids on silica gel-loaded paper. The distribution of 33P was as follows: 13.0y0 in phosphatidylglyccrol, 69.2% in phosphatidylethanolamine, 8.6% in cardiolipin, and 8.9% in unknown Compound X (Fig. I). There were no significant differences in the lipid composition from PA-l and PA-2. These values arc in agreement with values obtained previously with E. coli K-12 strains (21, 23).
Since the procedures used involve chloroform-methanol extraction of both cells and medium at each time point, the changes indicated above all involve conversion of phospholipid phosphodiester phosphate into non-lipid, water-soluble forms and not simply the loss of phosphate-containing lipid from the cells into the medium.
The cells fully labeled with V were carefully washed at 37" and resuspended at 37" in medium containing 3'Pi of known specific activity.
The rate of phospholipid breakdown (loss of 33P) and phospholipid synthesis (azP incorporation) was observed simultaneously by sampling at suitable time intervals, extracting, and fractionating on silica gel-loaded paper. Data from such an experiment on stringent and relaxed cells, in the presence and absence of a required amino acid, are given in Figs Relaxed cells, when deprived of a required amino acid degrade phosphatidylethanolamine at a very rapid rate (loss of 33P in Fig. 9) compared with growing cells (Fig. 10) or stringent cells deprived of a required amino acid (Fig. 11). It is also apparent (V + V, Fig. 9) that in relaxed cells starved for the required amino acid, the rate of 32P uptake only slightly exceeds the rate of 33P breakdown in phosphatidylethanolamine.
This observation is very striking in view of the known stability of phosphatidylethanolamine in growing cells under a variety of conditions (19,(29)(30)(31).
In more recent work, the latter group has found that a simultaneous downshift in aeration was imposed on the culture at the same time that these cells were deprived of the required amino acid. When the aeration (shake-rate) was kept constant (adequate for maximal growth) the results reported by Sokawa et al. (14) were 0btained.l We have verified these observations in our laboratory, observing a marked decrease in the rate of [%]acetate into lipid when the agitation of a rapidly shaken culture is greatly slowed at the same time that the [%]acetate is added. However, we find no major effect of aeration or the lack of it on the relative I'hosphatidylglycerol was also broken down at a rapid rate in t)he relaxed cells deprived of an essential amino acid (Fig. 12) 1 AcrtylLCo?l is synthesized endogenously by these organisms from the carbon source, and the intracellular specific activity of the acetyl-CoA actually used for lipid biosynthesis must depend on the relative amount of intracellular [T]acetyl-Co,4 and endogcnous acetylLCo9.
It seems possible that different conditions 2 Unpublished experiments.
of growth (or nongrowth) might alter this specific activity. Thus we employed radioactive phosphate in our subsequent esperiments since the above arguments about the intracellular specific activity seem less relevant for phosphate as a precursor of phospholipids (the cell must normally obtain all of its phosphorous from the medium).
As we will subsequently indicate, essentiall\ equivalent results (i.e. some net accumulation of phospholipid in relaxed cells deprived of a required amino acid) can be obtained by using exogenous [14C]acetate and radioisot'opic Pi (when tulnover is taken into consideration) and when net phospholipid synthesis is assayed directly by determining changes in lipid phosphate calorimetrically after ashing.

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The minimal rate of ['"C]aeetate incorlx)ration into lipid seen in stringent amino acid-starved cells may reflect a small amount of turnover or csrhange (see below), since direct measurement of phospholipid accumulation by assaying for phospholipid phosphate after ashing shows no accumulation in the stringent cells under Il~ese conditions. X comparison of the relative magnitude of llhospholipid accumulation with the magnitude of incorporation of radioisotopically labeled uracil into stable RNA in relaxed cells deprivttl of a required amino acid indicates a larger accumulation of RNA than of pl~ospholipid.
Stringent control of the accumulation of either metabolite is equally effective.
The marked incorporation of 32P into the phospholipids predominantly rc~lnx~scnts turnover with only some net synthesis in rc>lased cells drprived of a required amino acid, as shown by several lines of evidence.
(a) The relative amount of "Pi incorl)orated into phospholipid was much greater than the amount of [I"C]acetate incorporated into lipid.
(b) Quantitative assay of the lipid phosphate indicated no accumulation of phospholipid per ullit of ccl1 mass except in the relaxed amino acid-deprived cells;; the lipid phosphate accumulation in the latter cells was of the same relative order of magnitudt as the [Wlacetatc accumulation.
(c) Synthesis and turnover of the individual phospholipids from stringent and rclased cells mere simultaneously determined in a dual label experiment.
l'l~osl~l~at.idylcthanolamine in relased cells fully labeled from 33Pi and deprived of a required amino acid loses V at nearly the same rate as exogenous 32Pi is incorporated.
Little turnover is observed for this phospholipid in stringent, amino acid-deprived cells or in growing cells. Phosl)hatid\-lglycrrol a)nthe+ and breakdown occurs to the same cxtrnt in relas(d, amino acid-deprived cells and in growing cells. In btringent cells, phosphatidylglyccrol is broken down and re synthrsizcd but, it does not accumulate. Nearly one-half of the initial label in cardiolipin \\-as lost in the first 30 min when either rrlasetl or stringc,nt cells xere deprived of a required amino acid. C'artliolil)in ITas actively resynthesized in relaxed, amino aciddeprived cells.
This phospholipid is 11or111ally rather stable (19, 29-31) compared with cnl~diolipin or phosphatidylglycerol (I 9, 21, 29-31). Synthesis and breakdown of l)hosl)hatid\-letllaliolalilinc in the absence of a required amino acid the11 appears to be a direct consequrnce of a lesion in the rel gcnc as is RNA accumulation (l-4). The precisc scqu(Ancc of enzymatic events leading to the loss of phosphate from an\-of the phospholipids, indeed the biological significance of such a lxoccss to the bacterium, is still an open question (32-34).
Two rc>ccnt proporals hare been made which would permit one rel gene product to effect a number of metabolic processes. If the stringent cell 110 longer transported a source of carbon and cncrgy into the cell (13), all energy-requiring 1)roccsscs would cease. We have shown that amino acid deprivation of relaxed autl stringent ~11s has essentially identical effects on phospholipid mctnbolism, wllclher the carbon and energy source is glucose, succinatr, or ribose. Ribohc and ~1 g ucose are transported by energydependent, constitutive systems (35), whereas succinatc apparently enters via a facilitated diffusion (36). If stringent control wrre an effect 011 transport, it \vould have to affect all three of the above transport systems in a similar manner.
A decreased rate of glucose l~hoal~l~orglation was consistent n-it11 the i&x of elIerg\-limitation (37), but close csamination of the nucleotide content of the ccl1 as a general indicator of cncrglevel w-a< Ilot entirely coil&tent with that idea (g-12). Instead, an unusual nucleotide, guanosine tetraphosphatc, was discovered (38), proposed as an inhibitor of R?;.L synthesis (39) and pc'rhaps as t,he gcncral metabolic inhibitor,. i.e. the rel gent Ixotluct.
Recent work is now inconsistent with guanosinc tctraphosphale being the rel gene product (40-44), although it may l)lay some role in the stringent and relaxed control ~~l~c~~on~rt~oll.
OIW might even argue for the inch&n of DK;ldependent Rn'X transcription in this category, given the large gaps in ow knowledge of the mechanisms for the transcription of stable RN;\ or mRNA, or both, and the kno\vIl association of DNA with tllc membrane (48-50).
\yc ITill test this IWW hypothesis in 0111 laboratory shortly.